Sirtuins are an essential family of nicotinamide adenine dinucleotide (NAD)-dependent enzymes, conserved from bacteria to humans. Here we study the bacterial sirtuin CobB in E. coli. To demonstrate its highly conserved enzymatic activities and substrates, we used targeted mass spectrometry to quantify CobB levels, lipoylation levels, protein interactions, and the significance of its function in various nutrient conditions.

Experiment Description

The experiments were completed using MC4100 E. coli (wild type, ΔcobB, and CobB overexpression via an IPTG-inducible promoter), and analyzed in triplicate. Total area values were exported from Skyline and normalized by the average MS1 intensity per run calculated using RawMeat. (1) CobB protein levels were measured by SRM-MS using an Orbitrap Velos. (2) Site-specific lipoylation levels of proteins ODP2 and GCSH were measured in wild type and ΔcobB cells using an Orbitrap Velos. (3) Protein interactions with His-CobB or IgG were quantified by PRM-MS using a Q-Exactive. (4) Protein levels were measured using PRM-MS with a Q-Exactive after growing cells in media with different carbon sources.